FBTSA assays performed to measure binding of the mutated BRCA1 BRCT domains to ACC1-P, BACH1-P, CtiP-P, AB-1P and AB-2P.

FBTSA assay revealing that binding of the WT BRCA1 BRCT domains to ACC1-P, BACH1-P, CtiP-P, AB-1P and AB-2P induce a measurable increase in the BRCT thermostability that raises with peptide concentration.

Cells are inevitably challenged by low-level/endogenous stresses that do not arrest DNA replication. Here, in human primary cells, we discovered and characterized a noncanonical cellular response that is specific to nonblocking replication stress. Although this response generates reactive oxygen species (ROS), it induces a program that prevents the accumulation of premutagenic 8-oxoguanine in an adaptive way. Indeed, replication stress-induced ROS (RIR) activate FOXO1-controlled detoxification genes such as SEPP1, catalase, GPX1 , and SOD2 . Primary cells tightly control the production of RIR: They are excluded from the nucleus and are produced by the cellular NADPH oxidases DUOX1/DUOX2 , whose expression is controlled by NF-κB, which is activated by PARP1 upon replication stress. In parallel, inflammatory cytokine gene expression is induced through the NF-κB-PARP1 axis upon nonblocking replication stress. Increasing replication stress intensity accumulates DNA double-strand breaks and triggers the suppression of RIR by p53 and ATM. These data underline the fine-tuning of the cellular response to stress that protects genome stability maintenance, showing that primary cells adapt their responses to replication stress severity.

Isothermal Titration Calorimetry curves obtained by adding the different phosphopeptides at (A-B) 200 mM onto the WT BRCT domains at 20 mM and (C-D) 100 mM onto the WT BRCT domains at 10 mM .

The black bar corresponds to WT BRCA1, the blue bars to VUS of classes 1 and 2, the grey bars to VUS of class 3 and the red bars to VUS of classes 4 and 5. Bars boxed in green correspond to mutants that are defective in phosphopeptide-binding, as observed using fluorescence based thermal shift assays. HR- marks VUS that are HR-defective.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

Western blot analysis of the expression of BRCA1 and I-SceI measured after expression of either WT BRCA1 or VUS in the HR assays. BRCA1 was revealed using an anti-BRCA1 (mouse, ab16780, Abcam) and I-SceI with an anti-HA (mouse, sc-7392, Santa Cruz Biotechnology Inc.).

Sepiolite is a natural clay silicate that is widely used, including biomedical applications; notably sepiolite shows promising features for the transfer of biological macromolecules into mammalian cells. However, before its use, such an approach should address the efficiency of binding to biological macromolecules and cell toxicity. Because sepiolite spontaneously forms aggregates, its disaggregation can represent an important challenge for improving the suspension performance and the assembly with biological species. However, this can also influence the toxicity of sepiolite in mammalian cells. Here, a very pure commercial sepiolite (Pangel S9), which is present as a partially defibrillated clay mineral, is used to study the consequences of additional deagglomeration/dispersion through sonication. We analyzed the impact of extra sonication on the dispersion of sepiolite aggregates. Factors such as sonication time, sonicator power, and temperature are taken into account. With increasing sonication time, a decrease in aggregation is observed, as well as a decrease in the length of the nanofibers monitored by atomic force microscopy. Changes in the temperature and pH of the solution are also observed during the sonication process. Moreover, although the adsorption capacity of bovine serum albumin (BSA) protein on sepiolite is increased with sonication time, the DNA adsorption efficiency remains unaffected. Finally, sonication of sepiolite decreases the hemolytic activity in blood cells and the toxicity in two different human cell lines. These data show that extra sonication of deagglomerated sepiolite can further favor its interaction with some biomacromolecules (e.g., BSA), and, in parallel, decrease sepiolite toxicity in mammalian cells. Therefore, sonication represents an alluring procedure for future biomedical applications of sepiolite, even when using commercial defibrillated particles.

Size Exclusion Chromatography profiles obtained on BRCA1 BRCT domains free (red curve) or in complex with BACH1-P (purple), AB-1P (green), AB-2P (blue). The experiment was performed using a Superdex-75 10/300 GL column (GE Healthcare) pre-equilibrated with 50mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol and protease inhibitors (Roche).

Mutated BRCT domains fused to GST were expressed in E. coli and purified by affinity chromatography using glutathione beads. This figure shows a SDS-PAGE gel with samples from the bacterial pellet (P), supernatant (S) and the glutathione beads (G) after incubation with the supernatant and washing. VUS were classified into 3 groups as a function of the amount of (1) soluble and (2) purified protein obtained from bacterial cultures. Lanes corresponding to a typical insoluble fusion protein, a typical poorly soluble fusion protein and a typical fusion protein as soluble as the WT fusion protein are boxed in orange, grey and black, respectively (colors are as in Figure 4 excepted for mutations that cause aggregation during purification, which are in yellow on Figure 4 even if they displayed a "grey-like" profile at the expression and first affinity purification stages).

Supplementary table I: List of 78 BRCA1 BRCT missense VUS from the UMD-BRCA1 database. Bioinformatics in silico protein and 3D prediction scores, classification from BRCAShare (ex-UMD-BRCA1), BIC, kConfab and ClinVar databases. Supplementary table II: Classification of the BRCA1 VUS C1697R on the basis of multifactorial information Supplementary table III: Quantification of the percentage of fluorescence corresponding to nuclear BRCA1. The yes/no results extracted from this quantification to obtain Figure 3B were deduced from a statistical analysis using a t-test calculation.

Plot of the HR efficiencies measured after expression of either WT BRCA1 or VUS normalized to the HR efficiency after expression of WT BRCA1. The HR efficiencies are here plotted as a function of the position of the mutation in the BRCA1 sequence. Statistical significance was calculated using a one-side paired student t-test. To account for the multiple testing, the p-values were adjusted using the Benjamini-Horchberg method at a level a=0.05. HR efficiencies significantly different from the WT value are marked by asterisks. They are indicated by * if p < 0.05 and ** if p < 0.01. All 7 (likely) causal (noted LC or C) variants (in red) caused a significantly decreased HR efficiency whereas all 6 (likely) neutral (noted LN or N) variants (in blue) caused no significant HR difference. Twenty-four VUS of class 3 could not provide WT HR efficiency.

Introducción. Este trabajo se centró en la síntesis, caracterización físicoquímica, evalua- ción de su bioactividad y aplicaciones biomédicas de nuevos nanomateriales biohíbridos. La sepiolita es un silicato natural nanofibroso que presenta atractivas características como nanoportador para la liberación de ácidos nucleicos en células, entre otras aplicaciones in- novadoras en el área de las ciencias de la vida y de la biotecnología. Métodos. La caracteri- zación físico-química y evaluación biológica de los biohíbridos fue realizada usando proce- dimientos y técnicas novedosas, incluyendo entre otras AFM, TEM, microscopía confocal, TLVM, FTIR, Potencial Zeta, UV-vis, FACS, Western-blot, RT- qPCR. Resultados. Se obtuvieron nuevos biohíbridos mediante ensamblado de ácidos nucleicos con nanofibras de sepiolita. Se propusieron sus mecanismos de internalización y respuesta celular. Demostramos que la sepiolita es un nanoportador prometedor para la transferencia estable no-viral de ADN plasmídico en bacterias, células de mamíferos y humanas, con eficiencia optimizada. Debido a su capacidad de anclar varias biomoléculas, la sepiolita constituye también un nanoporta- dor para la vectorización simultánea de diferentes moléculas biológicas, incluyendo proteí- nas, nucleasas y anticuerpos. Adicionalmente, obtuvimos nuevos protocolos para aumentar significativamente la eficiencia de transformación bacterial, y para la extracción de ADN de bacterias, mediante métodos rápidos, seguros y económicos que no requieren la preparación de células competentes, representando una ventajosa alternativa a los costosos kits comerciales. En conclusión, la sepiolita es un producto de bajo costo, baja toxicidad y fluorescencia natural, con facilidad y conveniencia en métodos de síntesis de biohíbridos sepiolita/ADN, con posibilidad de aumentar su eficiencia de transfección, que junto a potenciales desarrollos futuros, representa una atractiva nanoplataforma para aplicaciones en nanomedicina y nanobiotecnología.

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.

Background Primary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms. Recently, variants in genes involved in DNA repair have been shown to cause POI. Notably, syndromic POI with Fanconi anaemia (FA) traits related to biallelic BRCA2 truncated variants has been reported. Here, we report a novel phenotype of isolated POI with a BRCA2 variant in a consanguineous Turkish family. Methods Exome sequencing (ES) was performed in the patient. We also performed functional studies, including a homologous recombination (HR) test, cell proliferation, radiation-induced RAD51 foci formation assays and chromosome breakage studies in primary and lymphoblastoid immortalised cells. The expression of BRCA2 in human foetal ovaries was studied. Results ES identified a homozygous missense c.8524C>T/p.R2842C-BRCA2 variant. BRCA2 defects induce cancer predisposition and FA. Remarkably, neither the patient nor her family exhibited somatic pathologies. The patient's cells showed intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation compared with controls and FA cells. R2842C-BRCA2 only partially complemented HR efficiency compared with wild type-BRCA2. BRCA2 is expressed in human foetal ovaries in pachytene stage oocytes, when meiotic HR occurs. Conclusion We describe the functional assessment of a homozygous hypomorphic BRCA2 variant in a patient with POI without cancer or FA trait. Our findings extend the phenotype of BRCA2 biallelic alterations to fully isolated POI. This study has a major impact on the management and genetic counselling of patients with POI.

The DNA damage response (DDR) interrupts cell cycle progression to restore genome integrity. However, unchallenged proliferating cells are continually exposed to endogenous stress, raising the question of a stress-threshold for DDR activation. Here, we identified a stress threshold below which primary human fibroblasts, activate a cell-autonomous response that not activates full DDR and not arrests cell cycle progression,. We characterized this “pre-DDR” response showing that it triggers the production of reactive oxygen species (ROS) by the NADPH oxidases DUOX1 and DUOX2, under the control of NF-κB and PARP1. Then, replication stress-induced ROS (RIR) activates the FOXO1 detoxifying pathway, preventing the nuclear accumulation of the pre-mutagenic 8-oxoGuanine lesion, upon endogenous as well as exogenous pro-oxidant stress. Increasing the replication stress severity above the threshold triggers the canonical DDR, leading to cell cycle progression arrest, but also to RIR suppression. These data reveal that cells adapt their response to stress severity, unveiling a tightly regulated ”pre-DDR” adaptive response that protects genome integrity without arresting cell cycle progression.### Competing Interest StatementThe authors have declared no competing interest.

We showed that sepiolite can transfer DNA into mammalian cells, opening alluring avenues for biothechnological and biomedical applications. Importantly, mammalian cells spontaneously internalize sepiolite, facilitating the spontaneous transport and delivery of bound molecules, but increasing the risks of potential toxicity. In addition, the fibrous nature of sepiolite raised health concerns about possible asbestos-like effects, especially because sepiolite can transfer DNA into bacteria through Yoshida effect. In spite that extrapolations from bacteria to mammalian cells could correspond to over-interpretations and the classification by IARC as non-hazardous and non-carcinogenic, we addressed here the response of human cells to interactions with sepiolite. We tested three classical cell responses to stress. We show that mammalian cells respond to sepiolite exposure, inducing the production of reactive oxygen species and the expression of inflammatory cytokines. This shows that cells detect sepiolite contamination and respond. Remarkably, sepiolite exposure did not alter the cell cycle distribution and triggers neither the DNA damage response program nor apoptosis, suggesting that it does not significantly assault the genetic material in mammalian cells. The potential toxicity of chronic versus transient exposure to sepiolite is discussed.

DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are frequently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. This slowing down, known as replicative stress, plays a central role in tumour development. Complex processes, which are not yet fully understood, are set up to respond to this stress. Certain nucleases, such as MRE11 and DNA2, degrade the neo-replicated DNA at the level of blocked forks, allowing the replication to restart. The interferon pathway is a defense mechanism against pathogens that detects the presence of foreign nucleic acids in the cytoplasm and activates the innate immune response. DNA fragments resulting from genomic DNA metabolism (repair, retrotransposition) can diffuse into the cytoplasm and activate this pathway. A pathological manifestation of this process is the Aicardi-Goutières syndrome, a rare disease characterized by chronic inflammation leading to neurodegenerative and developmental problems. In this encephalopathy, it has been suggested that DNA replication may generate cytosolic DNA fragments, but the mechanisms involved have not been characterized. SAMHD1 is frequently mutated in the Aicardi-Goutières syndrome as well as in some cancers, but its role in the etiology of these diseases was largely unknown. We show that cytosolic DNA accumulates in SAMHD1-deficient cells, particularly in the presence of replicative stress, activating the interferon response. SAMHD1 is important for DNA replication under normal conditions and for the processing of stopped forks, independent of its dNTPase activity. In addition, SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. When SAMHD1 is absent, degradation of neosynthesized DNA is inhibited, which prevents activation of the replication checkpoint and leads to failure to restart the replication forks. Resection of the replication forks is performed by an alternative mechanism which releases DNA fragments into the cytosol, activating the interferon response. The results obtained show, for the first time, a direct link between the response to replication stress and the production of interferons. These results have important implications for our understanding of the Aicardi-Goutières syndrome and cancers related to SAMHD1. For example, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments that trigger the inflammatory response in cells deficient for SAMHD1. We can therefore imagine that blocking the activity of these enzymes could decrease the production of DNA fragments and, ultimately, the activation of innate immunity in these cells. In addition, the interferon pathway plays an essential role in the therapeutic efficacy of irradiation and certain chemotherapeutic agents such as oxaliplatin. Modulating this response could therefore be of much wider interest in anti-tumour therapy.

Abstract BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. Implications: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection.

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.